CN106636043B - Method for producing protease - Google Patents
Method for producing protease Download PDFInfo
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- CN106636043B CN106636043B CN201611230292.3A CN201611230292A CN106636043B CN 106636043 B CN106636043 B CN 106636043B CN 201611230292 A CN201611230292 A CN 201611230292A CN 106636043 B CN106636043 B CN 106636043B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
Abstract
The invention relates to a method for preparing protease, which is characterized in that a protease inhibitor is continuously added into a culture medium in a specific time period from a logarithmic phase to a stationary phase of microbial growth, and the protease inhibitor can effectively inhibit the self-degradation of the protease. And the inventor surprisingly finds that the protease inhibitor is continuously added into the culture medium within a specific time period of microbial growth, the growth of the fermentation microbes is not inhibited, and the yield of the protease is greatly improved.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to a method for preparing protease.
Background
The enzyme preparation has been widely applied to the aspects of food, medicine, light industry, chemical industry, environmental protection, agriculture, energy and the like. The enzyme preparation is a substance with catalytic activity extracted from organic organisms or secreted by microorganisms, and can be produced by bacteria, filamentous fungi, yeast and the like mainly through submerged fermentation.
However, in the production of an enzyme preparation by fermentation, the enzyme preparation is easily degraded and has poor stability, resulting in a low yield, which is particularly the case when a protease is produced by fermentation. Most of proteases derived from microorganisms are difficult to accumulate in large-scale industrial production because proteases tend to be self-degraded. The inhibitor can inhibit the self-degradation of protease, but the traditional method can greatly inhibit the growth of fermentation microorganisms after the inhibitor is added, so that the overall yield is reduced, and the existing fermentation production process cannot be applied to the improvement of the yield of the protease. Another solution is a method for increasing the yield of protease by controlling the temperature of the fermentation process, but this method still cannot solve the problem that the yield of fermentation is limited due to the self-degradation of protease.
In summary, in the traditional process of producing protease by microbial fermentation, the reduction of the yield of protease caused by the self-degradation of protease is an urgent technical problem to be solved in the industry, and the problem becomes a bottleneck restricting the industrial production of protease.
Disclosure of Invention
Based on this, there is a need for a method for producing a protease that can solve the problem of self-degradation of the protease and thus improve the yield of the protease.
A method of preparing a protease, comprising the steps of:
fermentatively culturing a microorganism in a culture medium and adding a protease inhibitor to the culture medium continuously for a specified period of time during which the microorganism is growing, the specified period of time being a period of time from the log phase to the stationary phase of growth of the microorganism; and
the microorganism secretes to produce the protease.
In one embodiment, the specified time period lasts from 4h to 48h, the protease inhibitor is present in the culture medium at a final concentration of 0.1g/L to 5g/L, and the protease inhibitor is added to the culture medium at a rate of 0.002 g/(L.h) to 1.25 g/(L.h).
In one embodiment, the protease inhibitor is a boronic acid derivative.
In one embodiment, the protease inhibitor is selected from the group consisting of 4-formylphenylboronic acid, N-2-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid, thiophene-3-boronic acid, 3-acetamidophenylboronic acid, N-acyl-peptidylboronic acid, 4-methylthiophene-2-boronic acid, 2-fluoro-5- (methoxycarbonyl) phenylboronic acid, 5-ethylthiophene-2-boronic acid, 2-fluoro-4- (methoxycarbonyl) phenylboronic acid, 5-bromothiophene-2-boronic acid, 4- (1-piperidinylmethyl) phenylboronic acid, 4- (1-pyrrolidinylmethyl) phenylboronic acid, dibenzothiophenyl-1-boronic acid, and mixtures thereof, 2- (methoxycarbonyl) -4-methylbenzeneboronic acid, 2, 6-dichloro-3-methylbenzeneboronic acid, dibenzofuran-4-boronic acid, 2-isopropoxyphenylboronic acid, 2-fluoro-4-methylsulfonylboronic acid, furan-2-boronic acid, 3-fluoro-4-methylsulfonylboronic acid, 1-Boc-7-methoxyindole-2-boronic acid, furan-3-boronic acid, 4- (1-piperidinylsulfonyl) phenylboronic acid, 3-acetamidophenylboronic acid, 3-methoxythiophene-2-boronic acid, 2-methyl-4-methoxybenzeneboronic acid, 5-methyl-3-pyridineboronic acid, 5-n-propylthia-2-boronic acid, 2-methyl-4-methoxybenzeneboronic acid, 2-methyl-4-pyridineboronic acid, 2-methyl, 10-phenyl-9-anthraceneboronic acid, 3-bromothiopheneboronic acid, 5-methyl-6-fluoro-3-pyridineboronic acid, 3-bromothiophene-4-boronic acid, 2, 6-difluoro-3-pyridineboronic acid, 4-formylphenylboronic acid, 3-chloro-4-methoxycarbonylphenylboronic acid, phenylboronic acid, 2-chloro-4-methoxyphenylboronic acid, 5-ethylfuran-2-boronic acid, 3-methyl-4-chlorophenylboronic acid, diphenylboronic acid, 4- (cyclopropylsulfamoyl) phenylboronic acid, and 5-methyloxafuran-2-boronic acid.
In one embodiment, further comprising adding a protease stabilizing agent to the culture medium for a specified period of time during which the microorganism is growing.
In one embodiment, the protease stabilizing agent is selected from at least one of magnesium ions and calcium ions.
In one embodiment, the specific time period is 4 to 48 hours in duration, and the final concentration of at least one of the magnesium ion and the calcium ion in the medium is 0.005 to 0.1 mol/L.
In one embodiment, the microorganism is Bacillus subtilis, the addition of 4-formylphenylboronic acid to the culture medium is started 35 to 40 hours after the Bacillus subtilis is fermentatively cultured in the culture medium, the 4-formylphenylboronic acid is continuously added to the culture medium within a period of 5 to 20 hours, the final concentration of the 4-formylphenylboronic acid in the culture medium is 0.2 to 0.6g/L, and the rate of addition of the 4-formylphenylboronic acid to the culture medium is 0.01 g/(L.h) to 0.12 g/(L.h).
In one embodiment, further comprising adding a protease stabilizer selected from at least one of magnesium ions and calcium ions to the medium for a specified period of time during which the microorganism is growing, the final concentration of the at least one of magnesium ions and calcium ions in the medium being from 0.0125mol/L to 0.025 mol/L.
In one embodiment, the microorganism is pichia pastoris, 4-formylphenylboronic acid is added to the culture medium after the pichia pastoris is subjected to fermentation culture in the culture medium for 30h to 40h, the 4-formylphenylboronic acid is continuously added to the culture medium within a time period of 5h to 20h, the final concentration of the 4-formylphenylboronic acid in the culture medium is 0.2g/L to 0.6g/L, and the rate of adding the 4-formylphenylboronic acid to the culture medium is 0.01g/(L · h) to 0.12g/(L · h).
According to the method for preparing the protease, the protease inhibitor is continuously added into the culture medium in a specific time period from the logarithmic phase to the stationary phase of the growth of the microorganism, and the protease inhibitor can effectively inhibit the self-degradation of the protease. And the inventor surprisingly finds that the protease inhibitor is continuously added into the culture medium within a specific time period of microbial growth, the growth of the fermentation microbes is not inhibited, and the yield of the protease is greatly improved.
In addition, a protease stabilizer is continuously added into the culture medium in a specific time period of microbial growth, and the protease inhibitor is matched with the protease stabilizer, so that the yield of the protease can be further improved.
Drawings
FIG. 1 is a flow chart of a method for preparing a protease according to one embodiment;
FIG. 2 is a schematic diagram of the growth curve of Bacillus subtilis SCK6 in example 1;
FIG. 3 is a graph showing the growth curve of Pichia pastoris GS115 in example 1.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Referring to FIG. 1, a method for preparing a protease according to an embodiment includes the following steps S110 to S120.
S110, fermenting and culturing the microorganism in a culture medium, and continuously adding a protease inhibitor into the culture medium in a specific time period of microorganism growth, wherein the specific time period is a time period from a logarithmic phase to a stationary phase of microorganism growth.
Specifically, the medium used for the fermentative culture of the microorganism of the present invention means any conventional medium suitable for the growth of the microorganism, including a minimal medium and a complex medium. The Culture medium can be obtained by a method known in the art, which is commercially available from commercial suppliers, or by a method disclosed in the organizations such as China General Microbiological Culture Collection center (CGMCC), China Center for Type Culture Collection (CCTCC), American Type Culture Collection (ATCC), German Collection of microorganisms and cell cultures (DSMZ).
The method of producing a protease of the present invention can be used in industrial fermentative production of a protease on any scale, for example, in production of fermentation facilities on a scale up to 10L, 100L, 1000L, 10000L, or even 100000L. The fermentation culture process can adopt batch, repeated batch, fed batch and continuous fed fermentation methods. In one embodiment, the fermentation of the microorganism with Bacillus subtilis is a batch fermentation process. In another embodiment, the fermentation of the microorganism with pichia pastoris is a fed-batch fermentation process. The conditions of temperature, pH, etc. for fermentation culture can also be selected according to the characteristics of the specific cultured microorganism, and the fermentation culture can also comprise operations of microorganism inoculation, primary amplification culture, secondary amplification culture, etc., and the operations can be performed according to the general method for culturing the microorganism, which is not described herein again.
Specifically, the microorganism is also called protease producing strain, and the protease can be secreted and produced after the microorganism is fermented and cultured. The microorganisms in the present invention include bacteria and fungi. The bacterial protease producing bacteria are mainly Bacillus, examples of which include, but are not limited to, Bacillus subtilis, Bacillus licheniformis (Bacillus licheniformis), Bacillus brevis (Bacillus brevis), Bacillus cereus (Bacillus cereus), Bacillus thuringiensis (Bacillus thuringiensis), Bacillus megaterium (Bacillus megaterium), Bacillus mycoides (Bacillus mycoides). The above-mentioned strains can be obtained by researchers from Culture Collection institutions such as China General Microbiological Culture Collection Center (CGMCC), China Center for Type Culture Collection (CCTCC), American Type Culture Collection (ATCC), German Collection of microorganisms and cell cultures (DSMZ).
Fungal protease producing bacteria are primarily yeasts, examples of which include, but are not limited to, Torulopsis globulosa (Torulopsis) cells, Kluyveromyces (Kluyveromyces) cells, Hansenula polymorpha (Hansenula polymorpha) cells, Candida (Candida) cells, Schizosaccharomyces (Schizosaccharomyces) cells, and Pichia (Pichia) cells. The above-mentioned strains can be obtained by researchers from Culture Collection institutions such as China General Microbiological Culture Collection Center (CGMCC), China Center for Type Culture Collection (CCTCC), American Type Culture Collection (ATCC), German Collection of microorganisms and cell cultures (DSMZ).
Specifically, the growth conditions of the microorganisms are divided into different stages according to the growth rate and specific growth rate of the microorganisms in the fermentation culture process. The growth curve of a microorganism under a particular culture condition can be obtained by measuring the change in biomass over time of the microorganism under that condition. The growth curve can reflect the specific time of the microorganism in four growth stages, namely a delay stage, a log stage, a stable stage and a decay stage, and can reflect the rule that the cell activity changes along with the time. It should be noted that different species of microorganisms, different culture conditions and different culture strategies may cause changes in the growth curve. The biomass of the microorganisms during the fermentation can be determined by measuring the absorbance, OD, of the fermentation broth at a wavelength of 600nm600A value of (d); it is also possible to measure the wet weight of the cells in the fermentation broth. In one embodiment, the microorganism is tested for OD by fermentation with Bacillus subtilis600The value of (c). In another embodiment, fermentation of the microorganism with pichia pastoris measures the value of the wet weight of the cell.
Specifically, the logarithmic phase of growth refers to the stage of dividing the growth of the microorganism into different stages according to the growth rate and specific growth rate of the microorganism during the fermentation process, wherein the stage when the growth of the microorganism reaches the maximum specific growth rate is called the logarithmic growth phase.
Specifically, the stationary phase of growth refers to a phase in which the growth of microorganisms is divided into different phases according to the growth rate and specific growth rate of the microorganisms during fermentation, wherein the phase when the growth rate and death rate of the microorganisms are in dynamic equilibrium is called the stationary phase.
In this embodiment, the growth curve of the microorganism cultured under certain conditions can be determined by a preliminary experiment, and specific time points corresponding to the delay phase, log phase, stationary phase and decay phase of the growth of the microorganism can be determined, thereby determining the time point at which the addition of the protease inhibitor is started and the duration of the addition. Specifically, in the operation of continuously adding the protease inhibitor to the medium for a specific period of time during which the microorganism grows, the specific period of time is within a period from the logarithmic phase to the stationary phase of the growth of the microorganism. That is, a protease inhibitor is continuously added to the medium as a specific period of time arbitrarily cut from the point of time when the microorganism starts the logarithmic phase growth to the point of time when the microorganism finishes the stable growth. More specifically, the specific time period is from the end of the logarithmic phase to the early stage of the stationary phase of the growth of the microorganism.
In one embodiment, the time at which the specified time period begins is in the log phase of the microbial growth curve. In another embodiment, the specific time period begins at a time that is in the stationary phase of the microbial growth curve.
In one embodiment, the specified time period is from 4h to 48h in duration, the protease inhibitor is present in the culture medium at a final concentration of from 0.1g/L to 5g/L, and the protease inhibitor is added to the culture medium at a rate of from 0.002 g/(L.h) to 1.25 g/(L.h). Specifically, the protease inhibitor may be prepared in a solution form, and the protease inhibitor may be uniformly added dropwise to the culture medium over a specific period of time. In another embodiment, the specific time period has a duration of 5h to 45 h. In another embodiment, the specific time period has a duration of 10 to 36 hours. In another embodiment, the specific time period has a duration of 15h to 24 h. The protease inhibitor is prepared into a solution and can be uniformly dripped into the culture medium. The inventor finds that the protease inhibitor is continuously added from the logarithmic growth phase to the stationary growth phase of the microorganism, can effectively inhibit the self-degradation of the protease, can obviously obtain higher protease yield compared with the method of adding the protease inhibitor at one time at the same time, and cannot inhibit the growth of the microorganism.
Specifically, a protease inhibitor is a substance that can reversibly or irreversibly bind to the active center region of a protease to decrease or eliminate the protease activity, but does not usually cause protein denaturation. In this embodiment, the protease inhibitor is a boronic acid derivative, which can reduce the activity of the protease by competitive inhibition. The boron atom belongs to the second period in the element period, the organic boric acid is in sp2 hybridization form under normal conditions, when the organic boric acid reacts with water molecules, the lone pair electron of the oxygen atom in the water molecules enters the vacant orbit of the boron and releases a proton, and the organic boric acid is also changed into sp3 hybridization form from sp 2. Generally, the acidity of the phenyl boronic acids with different substituents varies greatly, and the pKa value ranges from 4.5 to 8.8. Under physiological pH conditions, the organic boric acid compound is easy to be hybridized from sp2 to sp3 hybridized negative ion form, the structure is very similar to the tetrahedral transition state of an enzyme catalysis substrate, and therefore, the organic boric acid compound can be used as an enzyme inhibitor. Since 1970, boronic acids were found to have enzymatic activity inhibiting properties, and scientists designed and synthesized a large number of these compounds, most of which were designed and synthesized as inhibitors of hydrolytic enzymes (e.g., protease inhibitors) and proteasome inhibitors. The protease inhibitor is selected from 4-formylphenylboronic acid, N-2-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid, thiophene-3-boronic acid, 3-acetamidophenylboronic acid, N-acyl-peptidyl boronic acid, 4-methylthiophene-2-boronic acid, 2-fluoro-5- (methoxycarbonyl) phenylboronic acid, 5-ethylthiophene-2-boronic acid, 2-fluoro-4- (methoxycarbonyl) phenylboronic acid, 5-bromothiophene-2-boronic acid, 4- (1-piperidinylmethyl) phenylboronic acid, 4- (1-pyrrolidinylmethyl) phenylboronic acid, dibenzothienyl-1-boronic acid, 2- (methoxycarbonyl) -4-methylbenzeneboronic acid, N-2-pyrazinecarbonyl-L-leucine boronic acid, N-methyl-2-phenylboronic acid, N-acetylamino-boronic, 2, 6-dichloro-3-methylphenylboronic acid, dibenzofuran-4-boronic acid, 2-isopropoxyphenylboronic acid, 2-fluoro-4-methylsulfonylboronic acid, furan-2-boronic acid, 3-fluoro-4-methylsulfonylboronic acid, 1-Boc-7-methoxyindole-2-boronic acid, furan-3-boronic acid, 4- (1-piperidinylsulfonyl) phenylboronic acid, 3-acetamidophenylboronic acid, 3-methoxythiophene-2-boronic acid, 2-methyl-4-methoxyphenylboronic acid, 5-methyl-3-pyridineboronic acid, 5-n-propylthia-2-boronic acid, 10-phenyl-9-anthraceneboronic acid, 2-methyl-4-pyridineboronic acid, 2-methyl-2-pyridineboronic acid, 2-fluoro-4-methylsulfonylboronic, 3-bromothiopheneboronic acid, 5-methyl-6-fluoro-3-pyridineboronic acid, 3-bromothiopheneboronic acid-4-boronic acid, 2, 6-difluoro-3-pyridineboronic acid, 4-formylphenylboronic acid, 3-chloro-4-methoxycarbonylphenylboronic acid, phenylboronic acid, 2-chloro-4-methoxyphenylboronic acid, 5-ethylfuran-2-boronic acid, 3-methyl-4-chlorophenylboronic acid, diphenylboronic acid, 4- (cyclopropylsulfamoyl) phenylboronic acid and 5-methyloxafuran-2-boronic acid.
In one embodiment, the protease inhibitor is present in the culture medium at a final concentration of 0.1g/L to 5 g/L.
In another embodiment, the protease inhibitor is present in the culture medium at a final concentration of 0.2g/L to 2.5 g/L.
In another embodiment, the protease inhibitor is present in the culture medium at a final concentration of 0.4g/L to 1.25 g/L.
In another embodiment, the protease inhibitor is present in the culture medium at a final concentration of 0.2g/L to 0.6 g/L.
Specifically, the adding rate of the protease inhibitor can be calculated according to the final concentration of the protease inhibitor in the culture medium and the continuous adding time.
In one embodiment, the microorganism is Bacillus, the protease inhibitor is 4-formylphenylboronic acid, and the final concentration of the protease inhibitor in the culture medium is from 0.2g/L to 0.6 g/L. 4-formylphenylboronic acid increases the yield of the protease of Bacillus. The increased protease production may, for example, refer to a regimen protease production after addition of a protease inhibitor that differs by 5% or more from a regimen protease production without addition of a protease inhibitor.
In one embodiment, further comprising adding a protease stabilizing agent to the medium for a specified period of time during which the microorganism is growing. The composition formed by matching the protease stabilizer and the protease inhibitor can more remarkably improve the yield of the protease.
Specifically, the protease stabilizer is a substance beneficial to maintain the normal structure of the protease, and the protease stabilizer may be at least one selected from magnesium ions and calcium ions. The source of calcium ions may be CaSO, for example4、CaCl2、Ca(H2PO4)2、CaHPO4、Ca(HCO3)2、CaCO3And Ca (NO)3)2At least one of (1). The source of magnesium ions may be, for example, MgSO4、MgCl2、Mg(H2PO4)2、MgHPO4、Mg(HCO3)2、MgCO3And Mg (NO)3)2At least one of (1).
Specifically, the final concentration of at least one of magnesium ions and calcium ions in the medium is 0.005mol/L to 0.1 mol/L. For example, the final concentration of at least one of magnesium ion and calcium ion in the medium is 0.075mol/L to 0.05 mol/L. Or, for example, at least one of magnesium ion and calcium ion is present in the medium at a final concentration of 0.01mol/L to 0.025 mol/L.
In one embodiment, magnesium ions may be separately added as a protease stabilizer, and the final concentration of magnesium ions in the medium is 0.005mol/L to 0.1 mol/L.
In another embodiment, calcium ions may be added separately as a protease stabilizer, the final concentration of calcium ions in the medium being 0.005mol/L to 0.1 mol/L.
In another embodiment, magnesium ions and calcium ions are added simultaneously as protease stabilizers, the final concentration of magnesium ions in the medium being 0.005mol/L to 0.1mol/L and the final concentration of calcium ions in the medium being 0.005mol/L to 0.1 mol/L.
Specifically, the protease inhibitor and the protease stabilizer may be added to the medium after mixing, or may be added to the medium separately. The inventors have found that when a protease is produced by fermentation using a microorganism, the production of the protease can be further improved by adding a calcium salt and/or a magnesium salt to the microorganism under the condition that a protease inhibitor is already added. Specifically, the increase in the protease production may mean, for example, that the difference between the protease production using a calcium salt or a magnesium salt as a stabilizer and the protease production without using the above stabilizer is 5% or more.
In one embodiment, the protease inhibitor and protease stabilizer form a composition that is added to the medium in an amount of 1g/L to 100 g/L. The composition comprises 0.1 to 10 weight percent of protease stabilizer and 0.01 to 10 weight percent of protease inhibitor.
In one embodiment, the microorganism is Bacillus subtilis, and the addition of 4-formylphenylboronic acid to the medium is started 35 to 40 hours after the Bacillus subtilis is fermentatively cultured in the medium, and the 4-formylphenylboronic acid is continuously added to the medium for a period of 5 to 20 hours. The final concentration of the 4-formylphenylboronic acid in the culture medium is 0.2 g/L-0.6 g/L. The 4-formylphenylboronic acid is added to the culture medium at a rate of 0.01 g/(L.h) to 0.12 g/(L.h). After the bacillus subtilis is fermented and cultured for 35-40 h and is at the end of logarithmic phase, adding a protease inhibitor into the culture medium, and continuously adding the protease inhibitor into the culture medium within a period of 5-20 h, wherein the 4-formylphenylboronic acid can inhibit the self-degradation of protease. The inventor unexpectedly finds that the protease inhibitor continuously added into the culture medium within a time period of 5-20 h after the bacillus subtilis is cultured for 35-40 h does not inhibit the growth of the bacillus subtilis, thereby greatly improving the yield of the protease.
More specifically, the method also comprises the step of adding a protease stabilizer after the bacillus subtilis is fermented and cultured for 35-40 h, wherein the protease stabilizer is selected from at least one of magnesium ions and calcium ions, and the final concentration of the at least one of the magnesium ions and the calcium ions in the culture medium is 0.0125 mol/L-0.025 mol/L. The 4-formylphenylboronic acid is matched with magnesium ions and/or calcium ions, so that the self-degradation of protease can be further inhibited, and the yield of the protease is improved.
In another embodiment, the microorganism is Pichia pastoris, the addition of 4-formylphenylboronic acid to the Pichia pastoris is started after 30 to 40 hours of fermentation culture in the culture medium, the 4-formylphenylboronic acid is continuously added to the culture medium within a period of 5 to 20 hours, and the final concentration of the 4-formylphenylboronic acid in the culture medium is 0.2 to 0.6 g/L. After fermentation culture of the pichia pastoris is carried out for 30-40 h, the pichia pastoris is at the initial stage of growth in a stable period, at the moment, a protease inhibitor is added into the culture medium, and the culture medium is continuously added into the culture medium within a time period of 5-20 h, so that the 4-formylphenylboronic acid can inhibit the self-degradation of the protease. And the inventors unexpectedly found that the protease inhibitor is continuously added into the culture medium within a time period of 5-20 h after the pichia pastoris is cultured for 30-40 h, the growth of the pichia pastoris is not inhibited, and thus the yield of the protease is greatly improved.
And (3) the microorganisms cultured in the fermentation processes of S120 and S110 secrete and produce the protease.
Proteases, also known as proteinases, proteolytic enzymes, peptidases or peptide hydrolases, are a generic term for a class of enzymes that can hydrolyze proteins or peptide chains. They are classified into endopeptidases and telopeptidases according to their hydrolysis. According to the nomenclature of NC-IUBMB, proteases belong to any enzyme of the EC3.4 enzyme family, and comprise thirteen subspecies, which are classified into serine proteases (S), cysteine proteases (C), aspartic proteases (A), metallo proteases (M), and unknown or unclassified proteases (U) based on their catalytic mechanisms, and later, glutamic proteases (G) and threonine proteases (T) were found, see in detail Handbook of Proteolytic enzymes, Neil D.Rawlings and Guy Salvesen, academic Press, 2013. There is no limitation on the origin of the protease produced by the present invention as long as the protease can be produced by secretion from a microorganism. Thus, the term protease includes not only natural proteases derived from organisms, but also any mutants and fragments or artificially synthesized proteases having protease activity.
Specifically, fermentation broth is obtained after fermenting the cultured microorganism, and the microorganism secretes and produces protease in the fermentation broth. The embodiment also comprises a step of recovering the protease, and the aspect of the invention is the recovery of the protease, namely, the downstream processing technology of fermentation. For example, it may be the pretreatment of the fermentation broth by flocculants (including chemical and biological flocculants); removing cells and residual nutrient substrate from the fermentation by filtration or centrifugation; concentrating the enzyme preparation by evaporation or ultrafiltration, and adding a stabilizing system to preserve the protease.
After obtaining the protease, the activity of the protease is also determined. The present invention may employ any assay for determining the activity of the recovered protease, for example, one or more assays employing one or more substrates comprising peptide bonds associated with the specificity of the protease, the temperature and pH of the assay employing a temperature and pH suitable for the protease. The yield of fermentation can be determined by measuring the activity of the recovered protease, and the yield of the method for producing the protease can be evaluated.
The method for preparing the protease continuously adds the protease inhibitor into the culture medium in a specific time period from the logarithmic phase to the stationary phase of the growth of the microorganism, and the protease inhibitor can effectively inhibit the self-degradation of the protease. And the inventor surprisingly finds that the protease inhibitor is continuously added into the culture medium within a specific time period of microbial growth, the growth of the fermentation microbes is not inhibited, and the yield of the protease is greatly improved. The method for preparing the protease can effectively solve the technical problem that the yield of the protease is reduced due to the self-degradation of the protease, and realizes the large-batch industrial production of the protease.
In addition, a protease stabilizer is continuously added into the culture medium in a specific time period of microbial growth, and the protease inhibitor is matched with the protease stabilizer, so that the yield of the protease can be further improved.
The following are specific examples.
Unless otherwise indicated, the experiments for formulating solutions and testing activity were performed at around 25 ℃.
The activity of the microorganism secreting the produced protease is determined by the following method in the following examples.
1. Solution preparation:
(1) preparation of Tris buffer: preparing Tris with the specification of 0.1M/L; 0.01mol/L CaCl2(ii) a 0.005% TritonX-100, pH 8.6 buffer. Specifically, 12.1g of Tris base was weighed and dissolved in 800mL of distilled water, and 1.1g of CaCl was added to the solution2Mixing with 50mg TritonX-100, adjusting pH to 8.6 with 6mol/L HCl (the concentration of commercial concentrated hydrochloric acid is 12mol/L generally, and diluting for use), and diluting to 1000mL with distilled water to obtain the final product.
(2) Preparation of stock substrate solution: a synthetic peptide N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SIGMA S7388) was prepared at 20mg/mL, using DMSO as a solvent. Adding 180mg of substrate into 9mL of DMSO, mixing and dissolving, putting into a centrifuge tube, wrapping with tin foil paper, and placing in a shade and dark place, wherein the substrate is not used up and discarded after one month.
(3) Working substrate solution: a synthetic peptide N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was prepared at a concentration of 1mg/mL, and the stock substrate was diluted 20-fold with Tris buffer, and prepared as needed, and the unused excess substrate was discarded.
(4) Preparation of protease solution (including control group): diluting the protease solution to be detected by about 250 times in the first step, then diluting the protease solution by about 125 times in the second step, firstly measuring the enzyme activity of the final dilution for the protease solution to be detected, and then measuring the enzyme solution obtained by the dilution in the first step if the difference between the enzyme activity and the control is larger. Specifically, 0.1 +/-0.05 g (to 0.1mg) of protease solution is accurately weighed in a 5mL centrifuge tube, then 0.1M Tris buffer solution is used for dissolving to 25g, stirring is carried out for 5 minutes, then 0.2 +/-0.05 g stock solution is accurately weighed in another 50mL centrifuge tube, then 0.1M Tris buffer solution is used for dissolving to 25g, and stirring is carried out for 5 minutes.
2. High throughput determination of protease activity using a microplate reader:
the total volume of a single reaction system is 200 mu L, each 180 mu L protease liquid to be detected is respectively added into a 96-hole plate, the 96-hole plate filled with the sample is placed in a sample bin of an enzyme-linked immunosorbent assay, the program is set to preserve heat at 37 ℃ for 5min, and a hatch door is automatically opened, so that the total volume of the single reaction system is 200 mu LWhen in use, a discharging gun is used to add 20 mu L of working substrate into each hole filled with the sample, the system is rapidly shaken for 20s and mixed evenly, and the speed (OD) of the released paranitroaniline is measured405) Reaction kinetic data were obtained. And finally, calculating the ratio of the reaction rate of the enzyme to be detected to the reaction rate of the reference enzyme, and calculating the enzyme activity.
Example 1
Production of protease by fermentation of Bacillus subtilis SCK6
1. Preparing a culture medium:
(1) primary slant seed medium (100 mL): 1.5g of agar powder; 5g of glucose; 1g of corn steep liquor powder; 1.5g of yeast powder; 0.75g of soybean protein; (NH)4)2SO41.5g；MgSO4·7H 20 0.4g；K2HPO40.2g；MnSO40.001g；FeSO40.001g；CaCl20.2 g; supplementing water to 100mL, heating, subpackaging 8mL in each test tube, sterilizing with high pressure steam, slightly inclining, cooling and solidifying for later use.
(2) Secondary liquid seed medium (100 mL): 5g of glucose; 1g of corn steep liquor powder; 1.5g of yeast powder; 0.75g of soybean protein; (NH)4)2SO41.5g；MgSO4·7H 20 0.4g；K2HPO40.2g；MnSO40.001g；FeSO40.001g；CaCl20.2 g; sterilizing with high pressure steam for later use.
(3) And a third-stage liquid seed culture medium: the same as the secondary liquid seed medium.
(4) Fermentation medium (1L): 100g of corn starch; barley flour 40 g; 30g of soybean hydrolysate; 10g of soybean protein; MgSO (MgSO)4·7H 20 4g；Na2HPO45g；MnSO40.01g；FeSO40.01g, adding 1g of α -amylase after preparation, boiling, stirring until the starch is liquefied, and filling into a fermentation tank for in-situ sterilization.
2. Inoculating and culturing
(1) Culturing first-level inclined plane seeds:
melting the strain SCK6 stored at-80 deg.C on ice, soaking a loop with inoculating loop, spreading on slant culture medium, and culturing in 37 deg.C incubator for 16 hr.
(2) Culturing secondary liquid seeds:
scraping thallus growing on slant culture medium with inoculating ring, inoculating into 1000mL conical flask containing liquid seed culture medium, loading liquid in 100mL, placing in rotary shaking table, rotating at 200rpm and 37 deg.C, culturing for 12 hr, determining cell growth concentration, and culturing to OD600Seeds were harvested at 0.7-0.9 days.
(3) Culturing the third-level liquid seed
Inoculating the second-level seeds into 5 2000mL conical flasks filled with liquid seed culture medium with the inoculation amount of 10%, placing the conical flasks with the liquid loading amount of 200mL into a rotary shaking table at the rotation speed of 200rpm and the temperature of 37 ℃, starting to respectively measure the cell growth concentration after culturing for 12h, and culturing to OD600Seeds were harvested at 0.7-0.9 days.
(4) Bacillus subtilis SCK6 fermentation culture secretion to produce protease
Inoculating the third-stage seeds into a 10L fermentation tank at an inoculation amount of 6%, wherein the liquid loading amount is 7L, and the air flow rate is controlled to be 0.8Nm3And h, automatically adding the defoaming agent, keeping the pressure of the tank body at 0.02MPa, fermenting at 35 ℃, keeping the culture time for 60h, and collecting 10mL of samples through a sampling tube every 2 h.
Controlling dissolved oxygen level: setting the stirring speed to be automatically controlled within the range of 25-45 percent, and adjusting the stirring speed by changing the stirring speed.
Stirring speed: the setting is linked with dissolved oxygen, and the adjusting range is 100 rpm-900 rpm.
According to the change of the biomass of the bacillus subtilis SCK6 with time under the condition, a growth curve is drawn as shown in figure 2, the time corresponding to a delay phase, a logarithmic phase, a stationary phase and a decay phase in the fermentation production is obtained, and then 10h, 20h, 35h and 40h after the fermentation is started are respectively used as the starting time for adding the protease inhibitor, wherein the protease inhibitor is 4-formylphenylboronic acid in the embodiment. And the yields were recorded without the addition of protease inhibitor.
The strategies for adding the protease inhibitor 4-formylphenylboronic acid are shown in table 1 below, each strategy was performed in a batch of experiments for producing protease by fermentation of bacillus, and the highest enzyme activity obtained under different strategies was compared to the ratio of the enzyme activity to that of the zymocyte without the protease inhibitor:
table 1: comparison of different protease inhibitor addition strategies with protease production without addition
Wherein, fermentation broth fb (fermentation broth), the start time of addition indicates the time point after how many hours the microorganism was cultured, and the duration time indicates that the protease inhibitor was continuously and uniformly added to the culture medium during the specified period of time. The final concentration (g/L) represents the final concentration of 4-formylphenylboronic acid in the medium. The yield was determined by the protease activity method described above. The enzyme activity ratio is the yield of the corresponding batch divided by the yield of the 0 th batch.
The experimental results of 1-36 batches of fermentation production in table 1 show that batches 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35 and 36 obtain higher enzyme yield and are significantly higher than the control batch 0 without protease inhibitor, which indicates that the yield of protease produced by fermentation can be significantly increased when protease inhibitor is added for 5-20 hours to a final concentration of 0.2-0.6 g/L after the bacillus SCK6 is cultured for 35 hours and 40 hours in late logarithmic production phase and stationary phase, i.e. fermentation culture.
Respectively collecting fermentation liquor with the fermentation time of 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 30h, 35h, 40h, 60h, 80h and 100h in the fermentation production experiments of 1-36 batches in table 1 to determine OD600The results of the growth curves obtained for batches 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35 and 36 are all in accordance with FIG. 2, indicating that the addition of 4-formylphenylboronic acid to the medium, which is continuously and uniformly maintained during the specified period of time during which the Bacillus subtilis SCK6 is growing, does not result in the addition of 4-formylphenylboronic acid to the mediumCan affect the growth of the bacillus subtilis SCK 6.
Example 2
Preparation of protease by fermentation of pichia pastoris GS115
1. Preparing a culture medium:
(1) pichia pastoris universal microelement (PTM1) stock solution (100 mL): FeSO4·7H2O，6.5g；ZnCl2，2.0g；CuSO4·5H2O，0.6g；MnSO4·H2O，0.3g；CoCl20.05 g; biotin, 0.03 g; na (Na)2MoO4·2H2O，0.02g；NaI，0.008g；H3BO30.002 g; concentrated H2SO4(98％，m/v)，0.5mL。
(2) Pichia pastoris universal microelement (PTM4) stock solution (100 mL): FeSO4·7H2O，2.2g；ZnCl2，0.7g；MnSO4·H2O，0.3g；CuSO4·5H2O，0.2g；CoCl2，0.05g；CaSO4·2H2O, 0.05 g; biotin, 0.02 g; na (Na)2MoO4·2H2O，0.02g；NaI，0.008g；H3BO30.002 g; after the substances are prepared and dissolved uniformly, concentrated H is carefully added2SO4(98％，m/v)，0.1mL。
(3) YPD medium (90 mL): 1g of yeast extract and 2g of peptone.
After autoclaving, 10mL of a sterile filter solution containing 2g of glucose was added.
(4) FM22 Medium (100 mL): KH (Perkin Elmer)2PO4，4.3g；(NH4)2SO4，0.5g；CaSO4·2H2O，0.1g；K2SO4，1.5g；MgSO4·7H2O, 1.2 g; glycerol, 4g and 0.25mL of PTM4 stock solution
(5) Fermenter glycerol feed medium (100 mL): 50g of glycerol and 1.2mL of PTM1 common trace element stock solution of pichia pastoris.
(6) Fermenter formaldehyde feed medium (100 mL): 98.8mL of methanol and 1.2mL of PTM1 Pichia pastoris universal trace element stock solution.
2. Inoculating and culturing
(1) Activation of production strains: inoculating the Pichia pastoris GS115 protease production strain preserved by ultralow temperature magnetic beads into 10mL YPD medium containing 26 mu g/mL bleomycin resistance to activate to OD600＝3.5，
(2) Culturing seeds: inoculating fresh activated culture solution into 375mL seed culture medium YPD at 3%, culturing at 29 deg.C for 20-24h in horizontal shaking table, and detecting OD600The culture was stopped until 4-5 was reached.
(3) Production of proteases by fermentation
Using FM22 culture medium as fermentation medium, loading 3.75L in 10L fermentation tank, sterilizing in situ, inoculating 375mL of harvested seed culture solution into the fermentation tank, controlling dissolved oxygen at 15-45% during fermentation, and ventilating 1.0Nm3And/h, setting the stirring speed to be automatically adjusted and linking with dissolved oxygen setting, wherein the change range is 100-900rpm, the temperature in the thallus growth stage is set to be 28 ℃, the temperature is set to be 26 ℃ after the thallus enters the induction stage, the pH is kept constant to be 5.5, only alkaline solution adjustment is needed under normal conditions, ammonia water is automatically added for alkali supplementation, defoaming agent is automatically added for defoaming setting, and 400mL of glycerol supplemented medium in a fermentation tank is added when carbon source depletion is detected.
Adding a formaldehyde feeding culture medium when the dissolved oxygen of the fermentation tank begins to rise to more than 15%, wherein the adding amount is about 0.5% (v/v) of the final concentration of the fermentation tank, and then beginning to feed the formaldehyde feeding culture medium at the speed of 6-14 mL/h for 8 h; and then controlling the adding speed of the formaldehyde feeding medium according to the change of dissolved oxygen to keep the dissolved oxygen between 15% and 45%, wherein the feeding speed is usually 3mL/L/h to 10mL/L/h until the fermentation is finished, and periodically sampling in the fermentation process to determine the biomass of the yeast, the protease activity of the fermentation supernatant and the content of protein.
According to the change of the biomass of the pichia pastoris with time under the condition, a growth curve is drawn and is shown in fig. 3, the time corresponding to a delay phase, a log phase, a stable phase and a decay phase in the fermentation production is obtained, 10h, 20h, 30h and 40h after the fermentation is started are respectively determined as the starting time for adding the protease inhibitor, and the protease inhibitor in the embodiment is 4-formylphenylboronic acid. And the yields were recorded without the addition of protease inhibitor.
The strategies for adding the protease inhibitor 4-formylphenylboronic acid are shown in table 2 below, each strategy was subjected to a batch fermentation experiment for producing protease by pichia pastoris fermentation, comparing the highest enzyme activity obtained under different strategies and the ratio thereof to the enzyme activity of the zymocyte without the protease inhibitor:
TABLE 2 comparison of different protease inhibitor addition strategies with protease production without addition
Fermentation broth fb (fermentation broth), the start time of addition indicates the time point after how many hours the microorganism was cultured, and the duration indicates the continuous and uniform addition of protease inhibitor to the medium during the specified period. The final concentration (g/L) represents the final concentration of 4-formylphenylboronic acid in the medium. The yield was determined by the protease activity method described above. The enzyme activity ratio is the yield of the corresponding batch divided by the yield of the 0 th batch.
The experimental results of fermentation production of batches 1-36 in table 2 show that batches 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35 and 36 obtain higher enzyme yield and are significantly higher than the control batch 0 without adding the protease inhibitor, which indicates that the yield of the protease produced by fermentation can be improved when the protease inhibitor is added for 5-20 hours to the final concentration of 0.2-0.6 g/L at the late stage of the logarithmic production phase and the stationary phase of pichia pastoris GS115, i.e., 30 hours and 40 hours after the beginning of fermentation.
In the fermentation production experiments of 1-36 batches in table 2, fermentation liquids with fermentation time of 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 30h, 40h, 60h, 80h, 100h, 120h and 140h are collected to determine the values of cell wet weight, and growth curves are drawn, so that the results of the growth curves of the batches 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35 and 36 are consistent with those in fig. 3, and it is shown that the continuous and uniform addition of 4-formylphenylboronic acid to the culture medium in a specific period of pichia pastoris GS115 growth does not affect the growth of pichia pastoris GS 115.
Example 3
Adding protease inhibitor to increase protease yield
The production strains and fermentation production conditions used for the fermentation of Bacillus were the same as in example 1.
The types of protease inhibitors added are shown in Table 3, each protease inhibitor was subjected to a batch fermentation experiment, the addition strategy was that after fermentation was carried out for 40h, protease inhibitors were initially added to the fermentation broth for 20h, the final concentration of each protease inhibitor was 0.4g/L, and the results of the fermentation enzyme yields and the ratio of the results to the enzyme yields (control) of the fermentation experiments without protease inhibitors are shown in Table 3 below
TABLE 3 comparison of protease production with and without protease inhibitor addition
The results in Table 3 show that the protease inhibitors shown in Table 3 were added at a constant rate for 20 hours after the Bacillus subtilis fermentation was carried out for 40 hours, so that the final concentration of each protease inhibitor reached 0.4g/L, which increased the protease yield.
Example 4
Effect of protease inhibitor and protease stabilizer combination addition on protease production by fermentation of Bacillus
The production strains and fermentation production conditions used for the fermentation of Bacillus were the same as in example 1.
The composition addition strategy was such that after 40 hours of fermentation, the addition of the protease inhibitor and stabilizer combination was started and continued for 20 hours at a constant rate, and the final concentration of each composition and the corresponding fermentation enzyme production results are shown in table 4 below:
TABLE 4 comparison of protease inhibitor and stabilizer composition with protease production without addition
From the results shown in table 4 above, it can be seen that the composition with the addition of protease inhibitors, calcium salts and/or magnesium salts can significantly improve the yield of the bacillus SCK6 fermentation protease, while the effect of the addition of calcium salts and/or magnesium salts alone on the yield of the enzyme is not significant.
Example 5
Effect of protease inhibitor and protease stabilizer composition addition on yield of protease produced by fermentation of Pichia pastoris GS115
The production strains and fermentation production conditions used for the fermentation of Pichia pastoris were the same as in example 2.
The strategy of adding the composition is that after fermentation is carried out for 30h, the composition of the protease inhibitor and the stabilizer is added, the constant speed is continued for 20h, and the final concentration of each composition and the corresponding fermentation enzyme yield result are shown in the following table
Table 5: comparison of protease production with and without addition of protease inhibitor and stabilizer composition
From the results shown in table 5 above, it can be seen that the composition added with the protease inhibitor, calcium salt and/or magnesium salt can significantly improve the yield of pichia pastoris GS115 fermentation protease, and the experimental results also show that the effect of only adding calcium salt and/or magnesium salt on the yield of pichia pastoris enzyme is not significant.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for preparing a protease, comprising the steps of:
fermenting and culturing a microorganism in a culture medium, wherein the microorganism is bacillus or yeast, and a protease inhibitor is continuously added into the culture medium within a specific time period of the growth of the microorganism, the protease inhibitor is a boric acid derivative, the specific time period is from the end of the logarithmic phase to the early stage of the stationary phase of the growth of the microorganism, the specific time period lasts for 5-20 h, and the final concentration of the protease inhibitor in the culture medium is 0.2-0.6 g/L; and
the microorganism secretes to produce the protease.
2. The method according to claim 1, wherein the protease inhibitor is added to the medium at a rate of 0.01 g/(L-h) to 0.12 g/(L-h).
3. The method for producing a protease according to claim 1, wherein the microorganism is Bacillus subtilis or Pichia pastoris.
4. The method for producing a protease according to claim 1 or 3, wherein the protease inhibitor is selected from the group consisting of 4-formylphenylboronic acid, N-2-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid, thiophene-3-boronic acid, 3-acetamidophenylboronic acid, N-acyl-peptidylboronic acid, 4-methylthiophene-2-boronic acid, 2-fluoro-5- (methoxycarbonyl) phenylboronic acid, 5-ethylthiophene-2-boronic acid, 2-fluoro-4- (methoxycarbonyl) phenylboronic acid, 5-bromothiophene-2-boronic acid, 4- (1-piperidinylmethyl) phenylboronic acid, 4- (1-pyrrolidinylmethyl) phenylboronic acid, a salt thereof, and a salt thereof, Dibenzothienyl-1-boronic acid, 2- (methoxycarbonyl) -4-methylphenylboronic acid, 2, 6-dichloro-3-methylphenylboronic acid, dibenzofuran-4-boronic acid, 2-isopropoxyphenylboronic acid, 2-fluoro-4-methylsulfonylboronic acid, furan-2-boronic acid, 3-fluoro-4-methylsulfonylboronic acid, 1-Boc-7-methoxyindole-2-boronic acid, furan-3-boronic acid, 4- (1-piperidinylsulfonyl) phenylboronic acid, 3-acetamidophenylboronic acid, 3-methoxythiophene-2-boronic acid, 2-methyl-4-methoxyphenylboronic acid, 5-methyl-3-pyridineboronic acid, 2-dichloro-3-methylphenylboronic acid, 2-fluoro-4-methylphenylboronic acid, 2-methyl-4-methylphenylboro, At least one of 5-n-propylthia-2-boronic acid, 10-phenyl-9-anthraceneboronic acid, 3-bromothiopheneboronic acid, 5-methyl-6-fluoro-3-pyridineboronic acid, 3-bromothiopheneboronic acid-4-boronic acid, 2, 6-difluoro-3-pyridineboronic acid, 4-formylphenylboronic acid, 3-chloro-4-methoxycarbonylphenylboronic acid, phenylboronic acid, 2-chloro-4-methoxyphenylboronic acid, 5-ethylfuran-2-boronic acid, 3-methyl-4-chlorophenylboronic acid, diphenylboronic acid, 4- (cyclopropylsulfamoyl) phenylboronic acid and 5-methyloxafuran-2-boronic acid.
5. The method of producing a protease according to claim 1, further comprising adding a protease stabilizer selected from at least one of magnesium ions and calcium ions to the medium for a specific period of time during which the microorganism grows.
6. The method for producing protease according to claim 5, wherein the protease stabilizer is selected from CaCl2And MgCl2At least one of (1).
7. The method according to claim 6, wherein the specified period of time has a duration of 20 hours, and the final concentration of at least one of the magnesium ion and the calcium ion in the medium is 0.0125mol/L to 0.025 mol/L.
8. The method for producing a protease according to claim 1, wherein the microorganism is Bacillus subtilis, wherein addition of 4-formylphenylboronic acid to the culture medium is started 35 to 40 hours after the Bacillus subtilis is fermentatively cultured in the culture medium, wherein the 4-formylphenylboronic acid is continuously added to the culture medium over a period of 5 to 20 hours, wherein the 4-formylphenylboronic acid has a final concentration of 0.2 to 0.6g/L in the culture medium, and wherein the 4-formylphenylboronic acid is added to the culture medium at a rate of 0.01 g/(L-h) to 0.12 g/(L-h).
9. The method of claim 8, further comprising adding a protease stabilizer selected from at least one of magnesium ions and calcium ions to the medium at a final concentration of 0.0125mol/L to 0.025mol/L in the medium, beginning 35h to 40h after the bacillus subtilis is fermentatively cultured in the medium.
10. The method for preparing protease according to claim 1, wherein the microorganism is Pichia pastoris, 4-formylphenylboronic acid starts to be added to the culture medium after 30-40 h of fermentation culture of the Pichia pastoris in the culture medium, the 4-formylphenylboronic acid is continuously added to the culture medium within a time period of 5-20 h, the final concentration of the 4-formylphenylboronic acid in the culture medium is 0.2-0.6 g/L, and the rate of adding the 4-formylphenylboronic acid to the culture medium is 0.01 g/(L-h) -0.12 g/(L-h).
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